tbuckley/cabot-search huggingface dataset:
MODL-09. SCAFFOLD-BASED (MATRIGELTM) 3D CULTURE TECHNIQUE OF GLIOBLASTOMA RECOVERS A PATIENT-LIKE IMMUNOSUPPRESSIVE PHENOTYPE
Authors: Frank Braun, Tanja Rothhammer-Hampl, Julia Lorenz, Sandra Pohl, Ayse-Nur Menevse, Arabell Vollmann-Zwerenz, Elisabeth Bumes, Maren Büttner, Saida Zoubaa, Martin Proescholdt, Nils Schmidt, Peter Hau, Philipp Beckhove, Beate Winner, Markus Riemenschneider
Journal: Neuro-Oncology (2023)

Abstract 3D (organoid) models in cancer research are considered the novel gold standard for in vitro experiments. Nevertheless, a variety of different protocols exits and the models are often used uncritically in lack of further validation. In this study, we aimed to characterize a scaffold-based (MatrigelTM) 3D glioma cell culture approach. We compared the 3D technique to conventional 2D culturing in its efficacy of generating models and in respect to its exact modeling properties. Models were generated from 62 patients with CNS tumors, most of them with glioblastomas, IDH-wildtype (GBM, n = 43). Pairs of 11 GBM models (2D and 3D) together with corresponding patient material were further characterized using bulk and single-cell RNA sequencing as well as 850k methylation analyses. Functional aspects of 3D tumor organoids were analyzed using a coculture system (Incucyte® SX5 live cell imager) with tumor infiltrating lymphocytes. Compared to conventional 2D culture the MatrigelTM-based 3D protocol was not superior in terms of the successful generation of in vitro models. However, NGS-based bulk and single-cell sequencing of matched pairs of 2D and 3D models showed an altered transcription of key immune regulatory genes in the 3D models, indicating the reoccurrence of an immunosuppressive phenotype. Changes included the presentation of different HLA surface molecules as well as cellular stressors. In the coculturing approach, lymphocyte-mediated cell killing appeared less effective in clearing 3D models than their 2D counterparts. IFN-γ release as well as live cell staining and proliferation analysis pointed towards an elevated resistance of 3D models. In conclusion, we found that the Scaffold-based (MatrigelTM) 3D culture technique depicts clinically highly important aspects of tumor-immune cell interactions, with the potential to explore immunotherapeutic approaches in an easily accessible in vitro system.
CNSC-07. USING CRISPR-CAS9 SCREENING TO IDENTIFY BIOMARKERS FOR MMF SENSITIVITY IN GLIOBLASTOMA MULTIFORME
Authors: Killian Buechler, Jack Shireman, Divya Ventarapragada, Graysen McManus, Jawad Fares, Cheol Park, Priya Kumthekar, Atique Ahmed
Journal: Neuro-Oncology (2023)

Abstract Glioblastoma (GBM) the most common malignant brain tumor with a median survival of 21 months. Recurrent GBM remains resistant to available therapies. Our phase I clinical trial investigates combining mycophenolate mofetil (MMF) and temozolomide (TMZ) to target de novo purine biosynthesis in GBM. Previously, our lab revealed that GBM cells upregulate purine biosynthesis and inhibit purine salvage through the ARL13B-IMPDH2 axis during TMZ therapy. Targeting IMPDH2 with MMF/TMZ extended survival in GBM patient-derived xenograft models. In preparation for Phase II of our clinical trial, we aim to identify a predictive biomarker for MMF+ TMZ therapy. In a CRISPR-Cas9-mediated knockout screen, we identified ten genes, including SLC1A5, showing enrichment (differential β score mean values &amp;gt; 60), indicating sensitivity to MMF/TMZ. SLC1A5 displays a wide distribution of expression in Glioviz patient datasets and encodes the glutamine transporter ASCT2, which is crucial for de novo purine biosynthesis. RNA-seq and protein expression analysis in six glioma cell lines confirmed that high SLC1A5 expression appears to correlate with sensitivity to MMF/TMZ (IC50 0.58-21.30 μM), while low expression led to resistance (IC50 73.43-1148 μM). Knockdown of SLC1A5 in a high-expression cell line demonstrated reduced sensitivity to MMF/TMZ (IC50 2.099 vs IC50 1007 μM). To further validate our findings, we plan to perform SLC1A5 overexpression in low-expression glioma cell lines. We hypothesize that GBM patients with higher SLC1A5 expression may respond favorably to MMF/TMZ therapy due to the reliance of their tumors on de novo purine biosynthesis. We aim to combine this data with analysis of our Phase I clinical samples to retrospectively establish SLC1A5 as a biomarker for MMF/TMZ sensitivity and investigate its role in tumorigenic purine metabolism. Identifying predictive markers will enhance personalized treatment strategies for GBM, offering hope for improved outcomes.
RTID-04. GLUGLIO - A PHASE IB/II RANDOMIZED DRUG REPURPOSING TRIAL OF GLUTAMATE SIGNALING INHIBITORS IN COMBINATION WITH CHEMORADIOTHERAPY IN PATIENTS WITH NEWLY DIAGNOSED GLIOBLASTOMA (NCT05664464)
Authors: Hans-Georg Wirsching, Patrick Roth, Angela Fischer, Andreas Hottinger, Thomas Hundsberger, Denis Migliorini, Adrian Ochsenbein, Heinz Laeubli, Katharina Seystahl, Lukas Imbach, Tibor Hortobagyi, Leonhard Held, Andrea Bink, Michael Weller
Journal: Neuro-Oncology (2023)

Abstract Glioblastoma synthesizes and secretes large quantities of the excitatory neurotransmitter glutamate, driving epilepsy, neuronal death, tumor growth and invasion. Several brain-penetrating drugs that have obtained clinical approval in other pathologic conditions can inhibit glutamate synthesis, secretion and signalling, respectively, including (i) the anti-seizure drug gabapentin, which inhibits the glutamate synthesis enzyme, branched chain amino acid transaminase 1, (ii) the anti-inflammatory drug sulfasalazine, which inhibits glutamate secretion by blocking the cystine-glutamate exchanger system Xc, and (iii) the cognitive enhancer memantine, which can prevent glutamate-driven, calcium-induced neuronal death and tumor cell invasion by blocking N-methyl-D-aspartate (NMDA) type glutamate receptors. The multi-centre, open-label, parallel-group, two-arm, 1:1 randomized, phase Ib/II trial GLUGLIO explores the efficacy of a triple-combination of daily oral gabapentin, sulfasalazine and memantine in combination with standard chemoradiotherapy compared to chemoradiotherapy alone. Dosing of either drug will be sought up to the maximum approved dose and will be reevaluated in an interim safety analysis after 20 patients have been randomized into the experimental arm. The primary endpoint is progression-free survival at 6 months (PFS-6). The sample size of N = 120 patients was calculated to detect an increase in PFS-6 from 50% to 70% with a power of 80% performing exploratory hypothesis testing at a one-sided significance level of 10%. Secondary endpoints include progression-free, overall and seizure-free survival, response rate, quality of life of patients and caregivers, symptom burden, cognitive functioning, tumor glutamate levels by magnetic resonance spectroscopy as well as anti-convulsant and steroid use. Surgical tissues as well as peripheral blood and electroencephalography recordings will also be sampled longitudinally for translational analyses. Recruitment is ongoing and foreseen to be completed by mid 2026. The simultaneous targeting of glutamate synthesis, secretion and signalling will clarify whether glutamate should be further explored as a clinical target in glioblastoma patients.
BIOM-51. IDENTIFICATION OF A LOW-FLIP ANGLE BASED CBV THRESHOLD FOR FRACTIONAL TUMOR BURDEN (FTB) MAPPING IN RECURRENT GLIOBLASTOMA
Authors: Aliya Anil, Ashley Stokes, Lea Alhilali, John Karis, Laura Bell, Jennifer Eschbacher, Leland Hu, Jerrold Boxerman, Kathleen Schmainda, C Quarles
Journal: Neuro-Oncology (2023)

Abstract INTRODUCTION Differentiation of glioblastoma recurrence from post treatment radiation effects (PTRE) would improve patient management. Dynamic susceptibility contrast (DSC) MRI can be used to generate fractional tumor burden (FTB) maps, via application of regional cerebral blood volume (RCBV) thresholds, to spatially differentiate recurrence from PTRE. FTB maps derived from a DSC-MRI protocol that includes a contrast agent preload in addition to the bolus injection and a moderate-flip angle (MFA) been histologically validated. Recently, a single-dose, low-flip angle (LFA) protocol was shown to provide RCBV measurements consistent with the MFA protocol and has been recommended as a consensus protocol. This study aims to identify the RCBV thresholds for the single-dose, LFA protocol that generates FTB maps, concordant with those obtained from the reference double-dose, MFA protocol. METHODS Two sets of DSC-MRI data were collected sequentially from 52 patients using a single-dose, low-flip angle (30o) protocol and a double-dose, moderate flip angle (60o) protocol using TR = 1.5s and TE = 30ms. Standardized RCBV maps (sRCBV) were obtained and co-registered with the post-contrast T1-weighted images using the FDA-approved clinical software, IB Neuro™ and IB Delta Suite™, respectively. The ground truth MFA-based FTB maps were computed using multiple sRCBV thresholds (1.0 and 1.75). An ROC analysis was conducted to identify the optimal, voxel-wise LFA sRCBV thresholds to produce FTB maps that best match the reference maps. RESULTS/ CONCLUSIONS The ROC analysis identified a lower LFA threshold of 1.0 to accurately distinguish PTRE from tumor recurrence and an upper threshold of 1.55 to differentiate aggressive tumor regions. The single-dose, LFA protocol eliminates variations in the contrast agent incubation time between the preload and bolus injection. This study provides motivation for the use of the single-dose LFA protocol in the management of patients with glioblastoma and is a reliable method for distinguishing tumor from PTRE.
MODL-50. CLONAL EXPANSION OF SPONTANEOUS AND INJECTED TUMORS IN SYNGENEIC MOUSE MODELS OF GLIOBLASTOMA
Authors: Courtney F Hall, Joanna Pyczek, Bo Young Ahn, Jennifer Chan, Sorana Morrissy
Journal: Neuro-Oncology (2023)

Abstract INTRODUCTION Glioblastoma (GBM) tumors are highly heterogenous and plastic. To better understand how tumors respond to treatment, it is important to use immunocompetent mouse models. We've created various mouse GBM models with common genetic mutations found in different subtypes of GBM, including classical, proneural, mesenchymal, and BRAF-driven. METHODS Using a combination of CRISPR and PiggyBac Transposon constructs, we injected plasmid mixtures into the ventricles of the mouse brain during the post-natal period. Electroporation was then used to enable the plasmids to enter nearby neural stem cells, which led to the spontaneous formation of tumors (spontaneous model). We collected tumor cells from these models and injected them into the adult mouse brain to create the injected mouse GBM model. Here we use spatial and single cell transcriptome profiling approaches to characterize these models at endpoint. Spatial transcriptome data was collected using the Visium 10X platform and tumor spots were deconvoluted from non-malignant spots using consensus non-negative matrix factorization. Copy number alterations (CNAs) were inferred using non-malignant spots and single-cell clusters as a reference. RESULTS We found that copy number states are largely homogenous within each sample, supporting an early population expansion in vivo. CNAs found in spontaneous tumors are also found in the matched injected tumors, which in turn harbour additional CNAs, suggesting that further selection occurs in vitro. Some of the CNAs include relevant cancer genes, suggesting the observed clonal expansions are potentially driven by additional somatic alterations that cooperate with the engineered driver mutations. Studying these models using orthogonal methods will allow us to discover alterations that synergize with the original drivers and delineate principles of GBM initiation and progression in an immune-competent environment.
MODL-03. AN EX VIVO PLATFORM BASED ON ORGANOTYPIC BRAIN SLICE CULTURE FOR INFORMING PERSONALIZED BRAIN CANCER THERAPY
Authors: Xiaopei Zhang, Breanna Mann, Noah Bell, Adebimpe Adefolaju, Morrent Thang, Rajaneekar Dasari, Krishna Kanchi, Alain Valdivia, Yang Yang, Andrew Buckley, Vivien Lettry, Carolyn Quinsey, Yasmeen Rauf, David Kram, Noah Cassidy, Cyrus Vaziri, David L Corcoran, Stephen Rego, Yuchao Jiang, Lee M Graves, Denise Dunn, Scott Floyd, Albert Baldwin, Shawn Hingtgen, Andrew B Satterlee
Journal: Neuro-Oncology (2023)

Abstract While the improved classification of CNS tumors is already enhancing the scientific rigor of clinical brain cancer treatment guidelines, the impact of these integrated diagnoses based on histopathological features or molecular insight on precision oncology medicine has yet to demonstrate widespread clinical benefit. Patient tumor-derived cancer models, including patient tumor-derived cell lines, organoids, and explants, have provided practical models based on a patient’s individual tumor for effective therapeutic drugs. Importantly, patient tumor-derived organoids have shown potential to guide personalized care and optimize clinical outcomes by functionally predicting patient response to antitumor drugs prior to in vivo treatment. Our team has built an organotypic brain slice culture (OBSC)-based platform and multi-parametric algorithm that allows for the ex vivo engraftment, rapid treatment, and systemic analysis of resected patient brain tumor tissue. This OBSC platform has successfully supported engraftment of each patient tumor tested to this point, including high- and low-grade adult and pediatric tumor tissue. The established patient tumor-derived organoids show the ability to activate the endogenous astrocytes within OBSCs, which have been reported in brain tumor. Whole exome sequencing analysis showed that patient tumor tissue engrafted onto OBSCs maintained a significant genetic resemblance to their parent tumor, while tumor tissue expanded in vitro displayed a distinctly different DNA profile. Additionally, our algorithm calculates dose-response relationships of both tumor kill and normal brain tissue toxicity, generating summarized drug sensitivity scores based on therapeutic window and allowing us to normalize response profiles across a panel of FDA-approved and exploratory agents. Summarized patient tumor scores after OBSC treatment revealed positive associations to clinical outcomes, suggesting the OBSC platform can provide rapid, accurate, functional testing to ultimately guide patient care.
MODL-07. <i>NF1</i> MUTATION IN OLIGODENDROCYTE PRECURSOR CELLS INDUCES PRENEOPLASTIC LESIONS IN THE BRAIN
Authors: Yuan Pan, Jared Hysinger, Belgin Yalçın, James Lennon, Preethi Raghavan, Nicole Schindler, Corina Anastasaki, Jit Chatterjee, Christopher Mount, Surya Nagaraja, Suzanne Scheaffer, Laura Attardi, David Gutmann, Michelle Monje
Journal: Neuro-Oncology (2023)

Abstract Oligodendrocyte precursor cells (OPCs) are progenitor cells that give rise to oligodendrocytes in the central nervous system throughout the lifespan. Transcriptomic and lineage tracing studies suggest that OPCs may be the putative cell of origin for many gliomas in children and adults. While children with the neurofibromatosis type 1 (NF1) cancer predisposition syndrome typically develop optic pathway and brainstem gliomas, as many as 50% of children develop gliomas in other brain regions using radiologic criteria. Relevant to these gliomas, discrete patches of hyperdense OPCs were found in the brains of Nf1+/- mice. The OPCs within the lesions display a transient increase in proliferation and hypercellularity. When the brains of mice carrying different heterozygous NF1 patient derived germline Nf1 gene mutations were examined, hyperdense OPC lesions were only seen in mice whose Nf1 mutations also predispose to optic gliomas. Mechanistically, monoallelic KRAS hyperactivation also results in similar OPC hyperdense lesions. Moreover, OPC-specific Nf1-heterozygous mice display similar lesions, while OPC-specific Nf1-knockout results in generalized OPC hyperdensity throughout the entire brain. Taken together, these data suggest the existence of a preneoplastic state conferred by Nf1 mutation in OPCs and establish a tractable model system to explore the transition to glioma in children with NF1.
MODL-10. SEVERELY IMMUNODEFICIENT NOG-EXL MICE ALLOW FOR HUMANIZATION AND DEVELOPMENT OF A HUMAN GLIOBLASTOMA-DERIVED TUMOR MICROENVIRONMENT
Authors: Zev Binder, Lamia Lamrani, Anthony Secreto, Nicholas Skuli, Moriah Jacobson, Charles-Antoine Assenmacher, Elinor Willis, Enrico Radaelli, Donald M O'Rourke, Cecile Alanio, MacLean P Nasrallah
Journal: Neuro-Oncology (2023)

Abstract Cellular immunotherapy has resulted in tremendous therapeutic advances across liquid tumors. However, for solid tumors, including glioblastoma (GBM), significant limitations still exist in successfully translating immune-based adoptive cell therapeutics from the bench to the clinic. The majority of preclinical in vivo GBM models either use human cell lines grown in immunodeficient animals or syngeneic tumors grown in immunocompetent animals. Both approaches lack critical immune components of the tumor microenvironment (TME), thus inherently limiting the ability to adequately model immunotherapies. Here, we demonstrate the ability of hematopoietic stem cells (HSCs) from a GBM patient to successfully engraft in the severely immunodeficient NOG-EXL mouse strain and generate a patient-derived immune system. Intraosseous injections of a low number (10,000) of CD34+ HSCs, obtained from a bone marrow draw from the patient at the time of tumor resection, resulted in an average of 20% human CD45+ cells in peripheral blood 17 weeks post-transplant. GBM organoids, established from the same patient, were implanted intracranially at Week 18 and allowed to grow for 5 weeks without significant clinical evidence of graft-vs-host disease or macrophage activation syndrome. Immunohistochemical and flow cytometry analyses of mouse brains, with and without tumors, revealed infiltration of human-derived, CD45+/CD33+ cells, in proximity to the tumor engraftment site. These results demonstrate the ability of patient-obtained CD34+ HSCs to engraft and form a TME in severely immunodeficient mice. Subsequent work will focus on the differences between autologous and allogeneic humanized tumor-bearing mice in the context of the originating tumor. Additional future work will focus on testing cellular therapies directed towards the relevant areas of the autologous model.
CNSC-08. PROTEOGENOMIC ANALYSIS OF LONGITUDINAL TRAJECTORY OF GLIOBLASTOMA EVOLUTION
Authors: Jong Bae Park, Antonio Iavarone
Journal: Neuro-Oncology (2023)

Abstract The evolutionary trajectory of glioblastoma after therapy is a multifaceted biological process that extends beyond discrete genetic alterations alone. Here, we performed a proteogenomic analysis of 123 longitudinal glioblastoma pairs, temporally separated by standard-of-care treatment. Integrative analyses identified a prevalence of a highly proliferative cellular state at diagnosis and its replacement by recurrent tumors sharing activation of neuronal differentiation and synaptogenicpathways. Proteomic and phosphoproteomic analyses revealed that the molecular transition from the proliferative to the neuronal state at recurrence is marked by coherent post-translational activation of the WNT/PCP signaling pathway and the BRAF protein kinase. Multi-omic analysis of the Patient-Derived Xenograft (PDX) model demonstrated similar patterns of evolutionary trajectory, marked by activation of neuronal signaling programs that were consistently observed in longitudinal patients. Inhibition of the BRAF kinase with small molecule inhibitors impaired both neuronal differentiation and migration capability of recurrent glioblastoma cells, which are the phenotypic hallmarks of glioblastoma progression after therapy. This work provides comprehensive insights into the biological mechanisms of glioblastoma evolution and treatment resistance and highlights new therapeutic opportunities to effectively counter them in the clinic.
TMIC-31. MULTI-PLATFORM GENOMIC LANDSCAPE OF INTRA-TUMORAL HYPOXIA IN GLIOBLASTOMA
Authors: Sheila Mansouri, Vikas Patil, Jeff Liu, Shreya Gandhi, Julio Sosa, Ron Wu, Kaviya Devaraja, Olivia Singh, Hafsah Ali, Shirin Karimi, Yasin Mamatjan, Sujun Chen, Anna Dvorkin-gheva, Shahbaz Khan, Hansen He, Thomas Kislinger, Federico Gaiti, Bradley Wouters, Gelareh Zadeh
Journal: Neuro-Oncology (2023)

Abstract Hypoxia is a prominent characteristic of aggressive tumors, including glioblastoma (GB), and is linked to therapy resistance. Understanding the molecular mechanisms underlying hypoxia-induced changes in neoplastic and non-neoplastic cells is crucial for developing targeted therapies. In this study, we took a comprehensive approach to investigate the genomic, epigenomic, and spatial transcriptomic profiles of intra-tumoral hypoxia in GB at bulk and single-cell levels. Our objectives were to identify a GB-specific hypoxia gene signature and uncover novel mechanisms of GB's response to hypoxia. Pimonidazole (PIMO), a hypoxia marker, was administered to 77 GB patients. We utilized immune-guided laser microdissection to extract DNA and perform methylome profiling of PIMO+ (hypoxic) and PIMO- regions. Visium 10X spatial transcriptomics was employed to identify differentially expressed genes in PIMO+ tumor regions. Through single-nuclear multi-omics profiling, we examined gene expression and chromatin accessibility of the hypoxic neoplastic and non-neoplastic cells at the single-cell level. The hypoxia transcriptome in GB exhibited general commonalities, in addition to unique features, compared to other reported hypoxia signatures. Some of these alterations were accompanied by changes at the DNA methylome level. Importantly, we found a heterogenous molecular landscape across PIMO+ regions within each sample. While a general transition to a MES-like state was observed, we found a mixture of cell states across the different PIMO+ regions. We also found distinct sub-hypoxic microenvironments harboring unique immune cell populations that overlapped with specific cell type states, cell cycle states, and deregulation of specific cellular pathways. These sub-regions were associated with specific anatomic features and displayed prognostic utility in GBM patient cohorts. Our single-cell multi-omics analysis further revealed intra-tumoral copy number aberrations and changes in chromatin accessibility associated with hypoxic cells. Our findings demonstrate that the hypoxic microenvironment in GB is heterogenous with unique targetable features that could lead to novel treatment modalities.